The origin of cardio-vascular disease in individuals often displays a complex background due to hereditary and age-related issues, but, in addition, also life-style dependent, so-called modifiable risk factors. When added together, these factors can be applied to account for the individual risk of developing cardio-vascular disease [1, 2]. One of these factors is the blood concentration of cholesterol. High serum levels of cholesterol, and in particular high levels of LDL-cholesterol (Low Density Lipoprotein), are epidemiologically strongly linked to an increase in the likelihood of developing of cardiovascular disease [3].
The classification of circulating cholesterol vehicle particles is derived from their settling under centrifugation. Consequently they are referred to as Very Low, Low, Intermediate and High Density Lipoproteins. The High Density Lipoprotein (HDL) is, contrary to the other particles, epidemiologically linked to a decrease in risk for developing cardio-vascular disease.
Chiefly two proteins are associated with the different particles, lipoprotein Al associated with HDL and lipoprotein B, associated with VLDL, LDL and IDL. As the proteins are found in discrete numbers for each of the particles, e.g. one copy of apolipoprotein B is found for each LDL-particle, it appears that determining the apolipoprotein blood concentration effectively yields a measure of the number of particles in circulation. Nowadays it is generally agreed that a treatment target should also include improving the lipoproteins and consequently it is becoming increasingly a routine matter in clinical practice to determine the lipoprotein concentrations as well as the blood cholesterol concentrations [4].
Cholesterol is a vital part of eukaryotic animal cells, with an important function in balancing the fluidity of the cellular membrane. In addition, cholesterol is the starting material for biosynthesis of e.g. vitamin D, cholic acids and various hormones [5]. Plant cells, on the other hand, rely on a whole family of chemically closely related phytosterols for similar functions. The chief difference between phytosterols and cholesterol resides in a substitution (commonly methyl- or ethyl-) on carbon 24 according to the sterol nomenclature [6].
Sterols belong to the category triterpenes. 4000 different triterpenes have yet to date been isolated; roughly 100 of these can be classified as phytosterols [7,8]. Interestingly, plant cells have developed a host of sterols, whereas animal cells have retained only one, cholesterol. The most prevalent of the phytosterols are the so-called 4-desmethyl-sterols, of which sitosterol, stigmasterol, campesterol, brassicasterol and avenosterol are the most prevalent. 4-methyl- and 4,4-dimethyl-sterols are usually less abundant and used as raw material for biosynthesis of 4-desmethyl-sterols [8]. Furthermore, the most commonly found sterols generally carry a double bond at carbon 5, subsequently they are referred to as Δ5-sterols. Fully saturated sterols, so-called stanols, can also be isolated from cereal sources, in particular rye and wheat. Phytosterols can be extracted from the oily parts of the plant, and hence also from the corresponding vegetable oils, such as corn, canola or tall oil [9].
As phytosterols are found in practically all plant materials typical western diets contain some 100 to 400 mg of phytosterols per day, the diet of vegetarians evidently being placed in the upper part of this interval [10]. In spite of the great chemical similarities between cholesterol and phytosterols a marked difference in the response to consumption of these molecules can be detected. Whereas cholesterol is efficiently absorbed from the intestine, phytosterols, on the other hand, are for all practical purposes hardly absorbed at all. Moreover, there are stark differences in response to the different molecules in the serum. Cholesterol is stored in the body, whereas phytosterols are usually rapidly expelled through the bile. The molecular differences result in the efficient blocking of cholesterol absorption by phytosterols in the intestine.
The cholesterol lowering properties of phytosterols were first documented in the early 50's with studies performed by Peterson on the cholesterol absorption in chickens fed phytosterol enriched fodder [11]. The understanding of the effect of phytosterols on animals and human subjects was improved with the work of Pollak, Best and Farquar and many others [12]. Typically, cholesterol lowering on the order of 10% is reached upon consumption of adequate doses of phytosterols with an almost exclusive decrease in the LDL-cholesterol serum levels; neither the HDL-cholesterol nor the triglyceride serum levels are significantly affected [13].
Cytellin, a cholesterol lowering preparation based on crystalline phytosterols was launched by Eli Lilly as early as 1955. The product had some severe flaws, such as a poor sensory profile and, more importantly, a substantial daily dosage required for clinical efficacy—in the range of tens of grams. Hence, the product was withdrawn from the market in 1982. In 1995, however, Raisio launched Benecol®, a yellow fatty spread based on a patented method for preparing stanols, hydrogenated sterols, esterified with fatty acids. By linking the stanol with fatty acids through esterification an increase in oil solubility by an order of magnitude was obtained. In addition, a considerable increase in stanol/sterol physiological efficacy was observed, where 10 to 15% of LDL-cholesterol reduction could be reached at daily dosages in the range of 1 to 3 grams. Since 1995 a number of similar products and technical solutions have been presented [9].
A number of theories have been presented to account for the cholesterol lowering mechanism of phytosterols, e.g. competition in micelles in the intestine, blocking of cellular receptors on the intestine wall and/or increase in reverse transport back into the intestinal lumen. Irrespective of the exact mechanism, the cholesterol lowering efficacy depends on the reduction of cholesterol absorption into the serum by approximately 50%. Importantly, the phytosterols themselves are poorly absorbed, around 5%, in comparison to around 60% for cholesterol. In addition, the rapid expulsion through the biliary route reduces the circulating phytosterol to less than 1% of the total sterol pool in a healthy individual. Moreover, phytosterols are not converted into cholesterol, or vice versa, in mammals, whereby the circulating phytosterols solely originate from consumed foods. However, neither crystalline phytosterols nor phytosterol esters are in fact directly available for cholesterol lowering purposes, rather the phytosterol molecules have to reside in a hydrolyzed and dissociated form to actively affect the cholesterol absorption process in the intestine [14].
Technical solutions for delivering phytosterol-enriched preparations can be subdivided into two main categories, viz.: i) chemically, i.e. by covalent bonding, altered phytosterols, e.g. esterified phytosterols or phytostanols; and ii) physico-chemically modified phytosterols, relying on weak physical interactions for maintaining the phytosterol molecules in a bioavailable, physiologically active form.
Due to their low melting point and enhanced oil solubility esterified phytosterols can usually, by emulsification with partially hydrolyzed lipids and cholic acids, form systems exhibiting rather small drop sizes and hence large surface area readily available for lipase activity in the intestine. In turn, the lipase hydrolyzes the covalent bond between the fatty acid and the phytosterol releasing phytosterols in monomolecular physiologically active form.
Common problems encountered with the application of esterified phytosterols are; shortened shelf life, which conveys requirements on cooled storage facilities. Moreover, the chemical process steps severely increase production costs and alter the composition and/or chemical structure from the natural composition and structure found in the original plant source.
Technical solutions relying on physico-chemical modification of the sterols include: i) dissolution of sterols, though only to levels of a few percent, in suitable oils, ii) generation of microcrystalline suspensions to guarantee a large available surface between the sterol crystals and surrounding food matrix, and iii) creation of an oil- or water-based emulsion by application of suitable emulsifying agents with or without addition of crystallization inhibitors.
The physiological efficiency of crystalline phytosterols is still very much doubtful, and these preparations are not likely to provide as efficient cholesterol lowering as e.g. a properly formulated emulsion, or phytostanol or phytosterol esters in a suitable oil-based environment.
Emulsion-based technical solutions tend to generate even more severe shelf-life problems than sterol or stanol esters. Moreover, emulsion-based sterol preparations tend to be inherently thermodynamically unstable, i.e. they are regular emulsions (not microemulsions), and as such rely on addition of suitable stabilizing agents for long term stability.
Food matrixes of choice include dairy products such as milk and yoghurts, juices and juice beverages, fatty spreads or cooking oils and bread. Enrichment of most of the mentioned food products with phytosterols involves dilution of the phytosterols to a relatively low concentration, usually well below 10%, in food products with limited shelf-life.
In summary, development of bioavailable, or physiologically active, preparations of phytosterols all have in common that a large available surface needs to be generated in the intestine in order for efficient molecular transport to the active sites to take place and facilitate lipase and esterase activity.
Due to the relatively large recommended dosage of 1 to 3 grams per day most phytosterol-enriched foods have dosages in the range of 20 to 40 grams of spread, or 100 to 200 ml of beverage. Hence, the prior art technical solutions are inadequate for preparing dry and, with respect to phytosterols, highly concentrated and physiologically highly active final products, such as tablets, or powders, due to long term stability problems, in some instances low physiological activity of the sterol preparation, or the physicochemical nature of the sterol preparation, e.g. the product is in liquid form.
Development of efficient preparations of phytosterols for producing compact and concentrated vehicles, such as tablets or capsules, in similarity to popular vitamin and mineral preparations would generate great benefits for the producer, as well as consumer of the phytosterol product. The phytosterol preparation would have to be efficiently prepared in order to maintain a highly bioavailable form of the phytosterol preparation even after final formulation and tablet manufacturing without loss of activity. Relatively few patents describe compositions and methods of preparation of phytosterol enriched tablets or pills.
US patent application No. 2006/0024352 describes the manufacturing of dietary supplements enriched with phytosterols in the form of tablets, capsules or suspension. The use of micronized or non-micronized powders of sterols in combination with common tablet excipients is mentioned, but no particular preparation of the phytosterols to enhance physiological activity is described.
US patent application No. 2006/0234948 describes phytosterol compositions containing lignans for the production of tablets containing between 50 to 400 mg of phytosterols. Phytosterols as well as other ingredients are only described as finely dispersed.
US patent application No. 2006/0251790 describes phytosterols recrystallized from triglycerides for tablet or pill applications.
International application WO/9956729 describes a composition containing a cholesterol lowering component; food containing such a composition, and a method to prepare the composition. In said method, the cholesterol lowering component is melted or dissolved in an organic phase under elevated temperature, and the obtained melt or solution, before crystallising or associating in other ways, is distributed in a matrix so that the cholesterol lowering component is stabilised mainly in monomolecular or low associated or “cluster” form. The product obtained is a solid, rubberlike or highly viscous mass.
International application WO/9956729 describes a composition comprising a mixture of a phytosterol and a surfactant as specified therein. Also described is the use of the composition as a pharmaceutical or in a food product.
Stanol-lecithin solutions in chloroform have been used for producing microparticles from a blend of phytostanols and lecithin by means of spray drying followed by granulation with polyvinylpyrrolidon and other excipients. Tablets obtained from this particular composition and preparation method display a statistically significant cholesterol lowering efficacy, however, the method presented seems difficult to apply in an industrial scale [15].
Towards this background it is evident that with respect to cholesterol lowering by use of phytosterol there still is a need for a formulation permitting to deliver a required daily dosage of phytosterol having an enhanced bioavailability, either as a pharmaceutical formulation or as a food product. One object of the present invention is to provide such a formulation.
The present invention is based on the finding that there are means of easily and efficiently preparing particles with high concentrations of phytosterols that do display a considerably increased physiological cholesterol lowering efficacy. The process of the invention involves a number of steps and provides a method suitably applying solely edible food-grade or the corresponding pharmaceutically acceptable ingredients.